Endothelial CaMKII as a regulator of eNOS activity and NO-mediated vasoreactivity

Figures

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  Fig 1. CaMKII is abundantly expressed in endothelium of different arterial beds. Immunofluorescence of CaMKII. Red: von Willebrand factor (endothelium); green: CaMKII; yellow: merged von Willebrand factor and CaMKII; blue: DAPI (nuclei). Arrows denote CaMKII expression. NC: negative control without primary antibody. Immunofluorescence in three arterial beds in human autopsy samples (Scale bars: 25 μm coronary and carotid artery, 5 μm aorta) and in three arterial beds in murine samples. (Scale bars: 50 μm).

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  Fig 2. Mouse models of endothelial CaMKII inhibition. A. Schematic of endo-CaMKIIN mouse model. B. Immunofluorescent detection of HA-tagged CaMKIIN in aorta from littermate controls (TekCre mice), CaMKIIN mice before TekCre recombination, and in endo-CaMKIIN mice (after Cre recombination). Red: CaMKIIN; green: eGFP (expressed before Cre recombination); blue: nuclei. C. Analysis of CaMKIIN mRNA levels by qRT-PCR. D. Schematic of endo-AC3-I mouse model. Expression of AC3-I occurs upon doxycycline (Dox) withdrawal. E. Immunofluorescent detection of eGFP-tagged AC3-I in littermate controls (eCdh5-tTA mice) and endo-AC3-I mice. Red: GFP; green: autofluorescence of elastin fibers; blue: nuclei. NC: negative control without primary antibody. F. Analysis of AC3-I mRNA levels by qRT-PCR. In B, E, Scale bars: 50 μm; arrows denote endothelium. Data in C and F are normalized to ARP and results expressed as mean ± SEM, n = 3 and 6 mice/group in C and F, respectively. *p<0.05 vs control.

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  Fig 3. Endothelial CaMKII inhibition does not affect baseline blood pressure or plasma NO levels. A, B. Basal systolic (A) and diastolic (B) blood pressures by tail cuff method in control and endo-CaMKIIN mice (n = 7–9 mice/group). C, D. Basal systolic (C) and diastolic (D) blood pressures by radiotelemetry in control and endo-AC3-I mice at indicated days after doxycycline withdrawal (n = 6–9 mice/group). E, F. Heart rate (E) and activity levels (F) in control and endo-AC3-I mice (n = 6–9 mice/group). G. Levels of total reducible NO (NOx), that includes nitrites and nitrates, in plasma of endo-AC3-I mice and littermate controls using a Sievers Nitric Oxide Analyzer NOA280i (n = 16–17 mice/group). H. Immunoblot for active CaMKII pThr-287 and total CaMKII in aortas of C57Bl/6 WT mice.

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  Fig 4. Inhibition of endothelial CaMKII does not affect vasorelaxation. A. Vascular relaxation in response to acetylcholine (ACh) in the presence or absence of the NOS inhibitor, L-NNA, was measured in pre-constricted aorta segments isolated from TekCre or endo-CaMKIIN mice (n = 11 TekCre and n = 9 endo-CaMKIIN mice/group). * p<0.05. B. Vascular relaxation in response to SNP in the presence or absence of L-NNA was measured in pre-constricted aorta segments from TekCre or endo-CaMKIIN mice (n = 10 TekCre and n = 8 endo-CaMKIIN mice/group). * p<0.05 vs. absence of L-NNA. C. Vascular relaxation in response to ACh in the presence or absence of L-NNA was measured in pre-constricted mesenteric arterial segments isolated from TekCre or endo-CaMKIIN mice (n = 7–8 TekCre and n = 7–8 endo-CaMKIIN mice/group). D. Vascular relaxation in response to SNP in the presence or absence of L-NNA was measured in pre-constricted mesenteric arterial segments isolated from control or endo-CaMKIIN mice (n = 7–8 TekCre and n = 6–8 endo-CaMKIIN mice/group). *p<0.05 vs. absence of L-NNA.

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  Fig 5. Ex vivo CaMKIIN delivery does not alter ACh-dependent vasorelaxation. A. eGFP expression in aortic rings of C57Bl/6 mice that were incubated with 20x107 particles of adenovirus expressing CaMKIIN (Ad5.CMV.IRES.CaMKIIN.HA) or no virus for 16–20 hr. Green: GFP, Blue: DAPI (nuclei). Arrow denotes endothelium. B, C. Vascular relaxation in response to Ach (B) or SNP (C) in aortic rings of C57Bl/6 mice after treatment with 20x107 particles of adenovirus expressing CaMKIIN or control adenovirus (Empty) for 16–20 hr. The same conditions were used in A, B, C. Some aortic rings were preincubated with 100 μM L-NAME for 10 min. D. Immunoblots for eNOS pSer-1179, eNOS, CaMKII pThr-497, CaMKII, GAPDH and HA were performed in aortic rings that were treated as indicated. E. Densitometric quantitation of the fold-increase in eNOS pSer-1179 relative to total eNOS. Data are the average of three independent experiments; *p<0.05 vs. Empty.

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  Fig 6. Bradykinin induces rapid CaMKII activation and Ca2+ influx in vitro. A. Representative immunoblots of phosphoThr-287 CaMKII (pCaMKII) and total CaMKII in 1 μM bradykinin (BK)-treated BAEC. B. Densitometric quantitation of the fold-increase in pCaMKII relative to total CaMKII. Data are the average of 4 independent experiments; *p<0.05 vs. 0 min time point. C. Representative tracings of BK-induced changes in Fura-2AM ratios in control HUVEC or HUVEC transfected with the CaMKII-specific inhibitory peptide autocamtide-2 (AC-2). Arrows indicate the time point when BK (1 μM) was administered. Each tracing represents a single cell. D. Areas under the curves from 5 sec before to 10 sec after BK administration. n = 4 independent experiments, *p<0.05 vs. control.

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  Fig 7. Acute inhibition of CaMKII attenuates eNOS activation in vitro. A. eNOS phosphorylation at Ser-1179 and Thr-497 following bradykinin (BK) treatment in BAEC infected with adenovirus expressing CaMKIIN or control adenovirus (Empty). Please note that in the eNOS immunoblot a molecular weight standard was loaded in an additional lane in between Empty and CaMKIIN treatment groups. B. Densitometric quantification of eNOS in BAEC infected with adenovirus expressing CaMKIIN or control adenovirus (Empty) (n = 8 independent experiments). C. Densitometric quantification of pSer-1179 immunoblots normalized total eNOS (n = 7 representative experiments); *p<0.05 vs. CaMKIIN at respective time point. D. Densitometric analyses of pThr-497 immunoblots normalized total eNOS (n = 7 representative experiments); *p<0.05 vs. CaMKIIN at respective time point. E. Calmodulin (CaM) binding to eNOS estimated by co-immunoprecipitation in cells stimulated with BK. F. Densitometric analyses of CaM binding to eNOS relative to total eNOS (n = 3 independent experiments), *p<0.05 vs. CaMKIIN at respective time point.

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  Fig 8. Inhibition of CaMKII abrogates bradykinin-induced NO production in vitro. A. NO-sensitive DAR-4M FM fluorescence in BAEC infected with adenovirus expressing CaMKIIN or control adenovirus (Empty) with and without treatment with 1μM bradykinin (BK). Green: eGFP; red: DAR-4M FM. As a positive control, cells were incubated with 10 μM of the NO donor DD1 after exposure to BK. B. Densitometric analyses of the intensity of the DAR-4M FM signal. Data are the average of 3 independent experiments. * p<0.05 vs. CaMKIIN.